Recombinant Salmonella heidelberg ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) from Salmonella heidelberg?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the F-type ATP synthase in Salmonella heidelberg. This protein consists of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The protein forms part of the membrane-embedded c-subunit ring structure that plays a crucial role in proton translocation across the membrane during ATP synthesis. In Salmonella, this protein contributes to energy metabolism and may influence bacterial survival under various environmental stresses .

How is recombinant Salmonella heidelberg atpE protein typically expressed?

Recombinant Salmonella heidelberg ATP synthase subunit c (atpE) protein is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The expression protocol generally involves:

• Cloning the atpE gene sequence into an appropriate expression vector

• Transforming competent E. coli cells with the construct

• Inducing protein expression under optimized conditions

• Lysing cells and purifying the protein using affinity chromatography

• Confirming protein identity via SDS-PAGE and Western blotting

• Lyophilizing the purified protein in a suitable buffer containing stabilizers like trehalose

This approach yields a recombinant protein that maintains structural integrity while providing the tag necessary for downstream applications.

What are the optimal storage conditions for recombinant Salmonella heidelberg atpE protein?

For optimal stability and functionality, recombinant Salmonella heidelberg atpE protein should be stored according to the following guidelines:

• Long-term storage: -20°C to -80°C in aliquots to prevent repeated freeze-thaw cycles

• Short-term working solutions: 4°C for up to one week

• Recommended storage buffer: Tris/PBS-based buffer containing 6% trehalose at pH 8.0

• Reconstitution: Deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol (5-50% final concentration) is advised for long-term storage

Repeated freeze-thaw cycles significantly reduce protein stability and functionality, so single-use aliquots are strongly recommended for experimental consistency.

What are the best methods for functional characterization of recombinant Salmonella heidelberg atpE protein?

Functional characterization of recombinant Salmonella heidelberg atpE protein can be approached through multiple complementary techniques:

• Membrane reconstitution assays: Incorporating the purified protein into liposomes to measure proton translocation activity

• ATPase activity assays: Measuring ATP hydrolysis rates in reconstituted systems

• Proton conductance measurements: Using pH-sensitive fluorescent dyes to monitor proton movement

• Site-directed mutagenesis: Identifying critical residues for function by creating point mutations

• Protein-protein interaction studies: Using pull-down assays to identify interaction partners within the ATP synthase complex

When conducting these experiments, it's essential to consider the membrane-embedded nature of the protein and its native lipid environment. Studies have shown that the c-subunit ring of ATP synthase may form or contribute to the mitochondrial permeability transition pore (mPTP), suggesting additional functions beyond ATP synthesis that may be relevant in pathogenic contexts .

How can researchers effectively purify recombinant Salmonella heidelberg atpE for structural studies?

For structural studies requiring high-purity atpE protein, the following optimized purification protocol is recommended:

Table 1: Optimized Purification Protocol for Structural Studies

This protocol accounts for the hydrophobic nature of the atpE protein, which requires careful handling with appropriate detergents to maintain its native conformation. For crystallographic studies, detergent screening is often necessary to identify conditions that promote crystal formation without destabilizing the protein structure.

What controls should be included when studying the immunogenicity of Salmonella heidelberg atpE protein?

When investigating the immunogenicity of Salmonella heidelberg atpE protein, the following controls should be incorporated:

• Negative controls:

  • Buffer-only treatment

  • Irrelevant recombinant protein with similar tag and expression system

  • Heat-denatured atpE protein to assess conformational epitope contributions

• Positive controls:

  • Known immunogenic Salmonella proteins (e.g., flagellin)

  • Commercial anti-Salmonella antibodies

  • Previously characterized epitopes from related ATP synthase components

• Specificity controls:

  • Cross-reactivity testing with atpE proteins from non-pathogenic bacteria

  • Epitope mapping to identify Salmonella-specific regions

  • Absorption studies with related proteins to remove cross-reactive antibodies

Recent studies on epitope mapping of recombinant Salmonella enterica proteins emphasize the importance of these controls for reliable immunogenicity assessment .

How does the structure-function relationship of atpE contribute to Salmonella heidelberg pathogenicity?

The structure-function relationship of atpE contributes to Salmonella heidelberg pathogenicity through several mechanisms:

• Energy production under stress conditions: ATP synthase function is critical for bacterial survival during host colonization. Studies of Salmonella Heidelberg outbreak isolates demonstrate that enhanced stress tolerance, particularly heat tolerance at temperatures relevant to poultry processing (56°C), correlates with outbreak potential . The atpE protein's structural integrity under stress conditions may maintain ATP production necessary for survival.

• Proton motive force maintenance: The c-subunit ring formed by atpE proteins controls proton flow across the membrane, which affects not only ATP synthesis but also membrane potential. This membrane potential is crucial for virulence factor secretion through type III secretion systems.

• Potential drug target: The unique structure of bacterial ATP synthase c-subunit makes it a potential target for antimicrobial development, particularly relevant given the increasing antibiotic resistance in Salmonella Heidelberg isolates .

Recent transcriptomic analyses of outbreak-associated Salmonella Heidelberg isolates revealed that exposure to heat stress increased expression of multidrug efflux and virulence genes , suggesting a coordinated response that potentially involves energy metabolism components like atpE.

What are the current challenges in using recombinant Salmonella heidelberg atpE protein for vaccine development?

Developing vaccines based on recombinant Salmonella heidelberg atpE protein faces several significant challenges:

• Membrane protein solubility: The hydrophobic nature of atpE makes it difficult to produce in soluble form without detergents, which can affect immunogenicity.

• Conservation across serovars: High sequence conservation of atpE across different Salmonella serovars may limit serovar-specific protection.

• Access to conformational epitopes: The native conformation of membrane-embedded atpE presents conformational epitopes that may be lost in recombinant proteins.

• Adjuvant requirements: As a small protein (79 amino acids), atpE may have limited immunogenicity without appropriate adjuvants.

• Cross-reactivity concerns: Potential cross-reactivity with host ATP synthase components could lead to autoimmune responses.

Research addressing these challenges has explored fusion protein approaches, novel adjuvant formulations, and epitope mapping to identify Salmonella-specific regions that could be incorporated into subunit vaccines . Additionally, combining atpE-based antigens with other Salmonella targets may provide broader protection against multiple serovars.

How can transcriptomic analysis inform the study of atpE expression in antibiotic-resistant Salmonella heidelberg strains?

Transcriptomic analysis offers valuable insights into atpE expression patterns in antibiotic-resistant Salmonella heidelberg strains:

Table 2: Transcriptomic Analysis Approaches for atpE Expression Studies

Research on outbreak-associated Salmonella Heidelberg isolates has demonstrated that transcriptomic analysis can identify differential gene expression patterns that correlate with enhanced stress tolerance . For atpE specifically, key research questions include:

• How does atpE expression change in response to antibiotics that target other cellular processes?

• Is atpE expression coordinated with multidrug efflux systems that contribute to antibiotic resistance?

• Does increased expression of ATP synthase components contribute to enhanced survival under antibiotic stress?

Transcriptomic studies have shown that outbreak-associated isolates may be "transcriptionally primed" to better survive processing stresses , suggesting that baseline expression of genes like atpE could serve as biomarkers for strains with enhanced survival capabilities.

What are the optimal conditions for studying protein-protein interactions involving Salmonella heidelberg atpE?

Studying protein-protein interactions involving the highly hydrophobic atpE protein requires specialized approaches:

• Membrane yeast two-hybrid (MYTH) system: More suitable than conventional Y2H for membrane proteins like atpE

• Cross-linking mass spectrometry (XL-MS): Recommended parameters:

  • Crosslinkers: DSS (for lysine residues) and zero-length EDC (for acidic residues)

  • Protein concentration: 1-2 mg/ml in 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.03% DDM

  • Crosslinking time: 30 minutes at room temperature

  • MS analysis: Orbitrap with HCD fragmentation

• Co-immunoprecipitation with intact ATP synthase complex:

  • Gentle solubilization with digitonin (1%) rather than harsher detergents

  • Pull-down using antibodies against other ATP synthase subunits

  • Western blot detection with anti-His antibodies for recombinant atpE

• Surface plasmon resonance (SPR):

  • Immobilization: Capture His-tagged atpE on Ni-NTA sensor chip

  • Running buffer: 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% DDM

  • Regeneration: 350 mM EDTA

When investigating interactions within the ATP synthase complex, it's important to consider that c-subunit ring assembly involves multiple copies of the atpE protein, which may complicate interpretation of interaction data.

How can researchers effectively study the role of atpE in Salmonella heidelberg heat tolerance?

To investigate the potential role of atpE in Salmonella heidelberg heat tolerance, researchers should consider the following methodological approach:

• Gene expression analysis:

  • Compare atpE expression levels between heat-tolerant and sensitive Salmonella Heidelberg isolates

  • Measure expression changes during heat shock (56°C, relevant to poultry processing)

  • Use both RNA-seq and protein-level validation (Western blot)

• Genetic manipulation:

  • Create atpE overexpression strains in heat-sensitive backgrounds

  • Generate atpE knockdown or conditional mutants (complete deletion may be lethal)

  • Complement mutants with wild-type and modified atpE variants

• Phenotypic assessment:

  • Heat tolerance assays at 56°C in stationary phase (where significant differences were observed)

  • Measurement of intracellular ATP levels before and after heat shock

  • Membrane potential assessment using fluorescent probes

• Structural analysis:

  • Compare atpE sequences between heat-tolerant and sensitive isolates

  • Model potential structural differences and their impact on c-ring stability

  • Identify potential structural adaptations that enhance thermal stability

Studies of outbreak-associated Salmonella Heidelberg isolates have demonstrated significantly increased heat tolerance in stationary phase at 56°C compared to reference strains , suggesting that ATP synthase components like atpE may contribute to this phenotype, potentially through maintaining energy production under stress conditions.

What approaches can be used to study the potential role of atpE in antibiotic resistance mechanisms in Salmonella heidelberg?

To investigate atpE's potential role in antibiotic resistance, researchers should employ a multi-faceted approach:

Table 3: Methodological Approaches for Studying atpE in Antibiotic Resistance

Research has shown that Salmonella Heidelberg isolates can exhibit resistance to multiple antibiotics, including ampicillin, chloramphenicol, tetracycline, streptomycin, and cefoxitin . The ATP synthase might contribute to resistance mechanisms through:

• Maintaining membrane potential required for efflux pump function

• Providing ATP necessary for energy-dependent drug efflux

• Adapting cellular metabolism to compensate for antibiotic-induced stress

Notably, horizontal gene transfer through conjugation is a primary mechanism for antibiotic resistance gene acquisition in Salmonella . Integrons, which are mobile genetic elements carrying antibiotic resistance genes, have been identified in Salmonella isolates and can be transferred via conjugation . While atpE itself may not be directly transferable through these mechanisms, its function may support the cellular adaptations necessary for expressing and utilizing acquired resistance determinants.

How might structural modifications of recombinant Salmonella heidelberg atpE improve its utility for research applications?

Strategic structural modifications of recombinant Salmonella heidelberg atpE can significantly enhance its research utility:

• Solubility enhancement:

  • Fusion with solubility-enhancing partners (MBP, SUMO, or Trx)

  • Introduction of strategic point mutations at hydrophobic residues

  • Creation of truncated constructs that maintain functional domains

• Stability improvements:

  • Introduction of disulfide bonds to stabilize tertiary structure

  • Removal of protease-sensitive sites

  • Surface entropy reduction to improve crystallizability

• Functional modifications:

  • Site-directed mutagenesis of key residues involved in proton translocation

  • Introduction of fluorescent protein tags for real-time localization studies

  • Addition of bioorthogonal chemistry handles for in situ labeling

• Immunological enhancements:

  • Fusion with immunogenic carriers to improve antibody production

  • Exposure of normally hidden epitopes

  • Modification of regions that may cross-react with host proteins

Each modification should be validated to ensure it doesn't disrupt the native function or structure of the protein. For instance, even small changes to the c-subunit structure could affect its ability to form the oligomeric ring essential for ATP synthase function.

What are the emerging approaches for studying the relationship between atpE and virulence in Salmonella heidelberg?

Cutting-edge approaches to investigate atpE's role in Salmonella heidelberg virulence include:

• CRISPR interference (CRISPRi) technology:

  • Allows titratable repression of atpE expression

  • Enables study of partial loss-of-function phenotypes

  • Can be used in infection models to assess virulence

• Single-cell analysis of metabolic states:

  • Correlates ATP synthase activity with virulence gene expression

  • Reveals population heterogeneity in metabolic adaptation

  • Identifies triggers for virulence expression

• Host-pathogen metabolic interaction modeling:

  • Maps how bacterial ATP production affects host metabolic responses

  • Predicts metabolic vulnerabilities during infection

  • Identifies potential intervention points

• In vivo imaging of ATP dynamics:

  • Uses genetically encoded ATP sensors

  • Tracks ATP fluctuations during infection process

  • Correlates energy production with virulence expression

Research has shown that some Salmonella Heidelberg outbreak isolates exhibited both enhanced heat tolerance and biofilm-forming ability , suggesting that energy metabolism components like atpE may contribute to multiple virulence-associated phenotypes. The transcriptomic analysis of outbreak-associated isolates revealed increased expression of virulence genes upon heat stress , pointing to a coordinated stress response that likely involves ATP synthase components.

How can systems biology approaches integrate atpE function into broader understanding of Salmonella heidelberg pathogenesis?

Systems biology offers powerful frameworks to contextualize atpE within Salmonella heidelberg pathogenesis:

Studies have demonstrated that outbreak-associated Salmonella Heidelberg isolates show transcriptional differences that may prime them to better survive processing stresses and potentially cause illness . A systems biology approach could reveal how ATP synthase components like atpE contribute to this enhanced fitness and identify potential intervention strategies that target energy metabolism pathways essential for pathogenesis.